User:Sophia C Zbesko/Notebook/Biology 210 at AU
16S Sequencing of Bacteria from Transect
Purpose: The purpose of this lab was to identify the different bacteria present in the transect by isolation and amplification of a single gene 16S.
Materials and Methods:
PCR Reaction--To prepare for PCR, samples of the bacteria in Colony A and B were transferred to 100μL of water in a sterile tube, incubating the tube at 100°C for 10 minutes in a heat block, and centrifuging the tube for 5 minutes at 13,400rpm. 20μL of the primer mixture was then added to the PCR tubes and mixed with a PCR bead. Following centrifugation, 5μL of supernatant from each sample was transferred to a new PCR tube. These tubes were then left in the PCR machine for one week.
Electrophoresis--Five μL of of each of the PCR products, from colony A and B bacteria, were prepared with 3μL of dye. The ladder of the agarose gel was prepared, followed by each sample being pipetted into one of the walls. The agarose gel was run and the results were viewed on a UV table.
Sequencing--The PCR product of colony A and B were sent off for sequencing.
16 S sequence for MB 45 (Colony B) NNNNNNNNNNGNNNNCTTNNNNNTGCAGTCGTACGNGTNGNCAGNGCNNNTNCNGATGCTGACGAGTGGCGAACGGGTGA GTAATACATCGGAACGTGCCTAGTAGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATGAGATCTACGGATGAA AGCAGGGGACCTTCGGGCCTTGTGCTACTAGAGCGGCTGATGGCAGATTAGGTAGTTGGTGGGGTAAAGGCTTACCAAGC CTGCGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA GTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCCTCGGGTTGTAAACTGC TTTTGTACGGAACGAAAAGCCTGGGGCTAATATCCCCGGGTCATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGT GCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTTGTAA GACAGTGGTGAAATCCCCGGGCTCAACCTGGGAACTGCCATTGTGACTGCANGGCTANAGTGCGGCAGAGAGNGGATGGA ATTCCGCGTGTANCANTGANATGCNTANATATGCGGAGNANCACCGATGGGCGAANNNNGTCCCCTGGGCCTGCACTGNN NCTCATGCACNNAAGCGTGGGGAGCAANANGANTNNNANNCTGGGTAGTCCACNCCCTNNCGATGTCAACTGNTTGTTGG NTNNNNNGTNANTCNNTAANNANNTANNNNTGAAGTTNACNNCNNNGGAGTACGNCCGGGNNGTTAGNANGNANNNNNNG GGNNTTGACCNCNCACCNCACTAGAGGNGGNGGTTGGGTTNNNNNNNNNNANNANNATAANCCCCCTNTCNCTTNNNANN ANNNNNGANNANANNGATAGNTGGTNGGGGGGNANNANNCNAACCGGNCCGCGGGGNCGGNGGGGACATCCCCCCCNNGG GTGNNTGNNACAAGNNNNGAAANCNCCNCANCCGNNGGGGCCNNNNTNGCNGNNGNNNNNNN
Colony B: 95% match for Comamonas testosteroni
16S for MB 46 (Colony A) NNNNNNNNNNNNNNNNNNNANNNTGCAGTCGNNCGGGNGTAGCNCGNTANNGCTCTCTNATTCGCGGCGGACGGGTGAGT AATGCCTANGAATCTGCCTGGTANTGGGGGACAACGTCTCCAAAGGGACGCTAATACCGCAAACGTCCTACGGGAGAAAG CGGGGGACCTTCGGGCCTTGCGCTATCGNATGANCCTAGGTCGGATTANCTAGTTGGTGAGGTAATGGCTCACCAAGGCC ACGATCCGTAACTGGTCTGAGAGGATGATCATTCACACTGGAACTGAGACACGGTCCNGACTCCTACGGGAGGCAGCAGT GGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTT TAAGTTGGGAGGAAGGGCAGTAACTTAATACGTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGC CAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTANGTGGTTTGTTAANT TGNATGTGAAANCCCCGGGCTCAACCTGGGAACTGCATTCNNAACTGACAAGCTANAGTATGGTANAGGGTGGTGNAATT TCCTGTGTAGCNGTGAAATGCNNNNNATNNGAAGGAACACCNGTGNCNAAGGCGACCACCTGNACTGATACTNACACTGA NNTGCGAAAGCGTGGGGANCNACANGATTANATNNCCTGNTACTCCACNNCNNTANACNATNTNNNCTANCCNTTTNGAN CNCTTGAGCTGNNNANTGNCGCANCTCACNCATTAANTTTNACCNNCCTNNNNTAGTACNGGCNCNNCGNTANGNNNTCN NNNGGANTNNNNNNNNCNGNCNNAANCANNNGANCTGGTGGTTTNNTTCNNNNNNANANNNANAACNNTACNNNNCCCNN NGNNTNNNNNNNCNNNNNNNNNNNNNNTTNNGGCGNNNNNNNNNNNNNNNNGNNNCNNNANNNNNGNNNNNNNNNNNNTN NNNNNNNNTGNANNNNNNNNNNNNNNNNNNNNANNNNNNN
Colony A is 93% match for both Pseudomonas mohnii and Pseudomonas putida
Conclusions Both of the sequencing had poor quality to them, but information aout each bacterium was still provied. The MB46 16S sequencing that resulted in Pseudomonas mohnii and Pseudomonas putida, basically corresponds to the prior observations done in the lab a few weeks back. This bacteria is a soil bacterium, that's gram negative (does not have a thick layer of peptidoglycan), and has a bacillus shape. In the prior observations, it was thought that this bacterium is spirillum shaped instead of bacillus. This could be due to not being able to focus well enough during the microscopic observations. On the other hand, the MB45 that resulted in Comammonas Testosteroni, is gram negative and bacillus shaped. This was not the same results conducted in lab. In lab, it was thought that the bactium was gram positive and had a coccus shape. The error came from the inability to use fine microscopes and/or improper staining which lead to variability in the results.
4.7.15 Good conclusions SK
Zebrafish and Embryonic Development: Lab 6 from February 19 to March 4, 2015
Purpose: The purpose of this lab was to see how environmental conditions can affect embryonic development, using the Zebrafish as the model organism.
Materials and Methods: First, a published paper on the effect of development in Zebrafish with Nicotine was ready to be developing an experiment to be conducted. Two petri dishes were labeled and used, one as a control (20 mL of deerpark water) and the other as the treatment (20 mL of 25 mg/L of Nictone). A total of 20 embryos was transferred to each petri dish with a pipette.
Data Observations: On day 0, the day of setting up the experiment, all the embryos were in their zygote stage. The tables below provide the data collected throughout the whole experiment.
Table 1: Control
|Control||Day 0 (2/19)||Day 1 (2/20)||Day 4 (2/23)||Day 7 (2/26)||Day 11 (3/2)||Day 13 (3/4)|
|# of Dead Eggs||0||0||4||1||0||0|
|# of Live Eggs||20||20||8||0||0||0|
|# of Live Hatchings||0||0||5||10||9||9|
|# of Dead Hatchlings||0||0||0||1||1||0|
|Stages of Development||Zygote||Zygote||8-->25-somites, 5--> high spec||10--> protuding mouth||-||0|
|Degree of Body/Tail Pigmentation||-||-||-||-||yellow, with black dots, straight||bits of yellow by head, blacks spots down body, striaght|
|tail= 28.7 5μm body=41.25 μm|
|Eyes/Eye Movement||-||-||-||-||strong, fast||rapid eye movement, eyes= 2.5 μm|
|Heart Rate||-||-||-||-||-||56 bpm|
|General Movement||-||-||-||-||jittery movement, fast reaction time (1sec)||constantly moving, fast reaction time (1sec)|
|Feces||-||-||-||-||mild, few piles (5)||4 piles|
Table 2: Treated
|Treated with Nicotine||Day 0 (2/19)||Day 1 (2/20)||Day 5 (2/24)||Day 7 (2/26)||Day 11 (3/2)||Day 13 (3/4)|
|# of Dead Eggs||0||6||5||0||0||0|
|# of Live Eggs||20||20 (added 6)||1||1||0||0|
|# of Live Hatchings||0||0||10||9||8||5|
|# of Dead Hatchlings||0||0||0||0||0||0|
|Stages of Development||Zygote||17-somites||1--> 21 somites, 10--> high spec||1-->25-somite 9--> protuding mouth||0||3|
|Degree of Body/Tail Pigmentation||-||-||-||-||either much more yellow w/ black dots or not yellow with black dots||body bent, minimum yellow,most clear with black dots down back|
|body was bent||tail=25 μm body= 40 μm|
|Eyes/Eye Movement||-||-||-||-||slow, delayed||no eye movement, eyes=2.75 μm|
|Heart Rate||-||-||-||-||-||60 bpm|
|General Movement||-||-||-||-||float around, very slow reaction time (3 sec)||floating, not much movement, slow reaction time(3), one was crazy|
|Feces||-||-||-||-||Lots of piles (20)||lots of piles (25)|
Conclusions and Future Directions: Past research indicated that placing zebrafish embryos in a nicotine solution would negatively impact their survival rate as well as their response time, eye diameter, tail length, and heart rate. In accordance to past experiments, the zebrafish treated with the nicotine solution had a higher mortality rate and reductions in other physical traits crucial to their survival.
4.7.15 Could have included more detailed methods. SK
2.20.15 Very good lab book entry. Nice descriptions of dismantling the Berlese funnel, but could include some details of its setup. Good food web, I especially like the pictures! SK
Invertebrates: Lab 5 on February 12, 2015
Purpose: The purpose of this lab was to characterize different invertebrates and vertebrates identified in the transect and understand their importance. It was also to understand how simple systems evolved into more complex systems.
Materials and Methods: To observe the invertebrates that lived in the transect, the Berlese Funnel, from last week, was disassembled from the conical tube with ethanol. One petri dish contained the first 15 mL of the ethanol and collected matter, while the second contained held the remaining 25 mL of ethanol and matter. The petri dishes were then examined under a dissection microscope where a key was used to identify the specific invertebrates. The key determines the specific species by the characteristics of the invertebrate such as its body parts, segments, and whether antennas were present.
Data Observations: There were three invertebrates analyzed and observed from the Berlese Funnel. All of the found in vertebrates identified as arthropods, and only two had the class of insect. There was a mite, a type of arachnid (possibly a Homoptera Hopper), and a beetle. The mite was the smallest and the beetle was the largest, which can be seen in Table 1.
Table 1: Invertebrates
No vertebrates were observed directly from the transect. Table 2 displays vertebrates that have been seen on American Univeristy's campus and would possibly inhabit a marsh transect. The Sora rail is common bird species who inhabits U.S. Marshlands, including those in the DC area (Parr, 2005. The American crow, brown rat, and northern raccoon have all been seen on American University;s campus, even though they may have not been directly seen in the marsh transect. The brown rat and northern raccoon are also known to inhabit marshes (Armitage, 2004; Fox, 2001). Table 2 also shows the abiotic and biotic features of the transect that would benefit each species.
Table 2: Vertebrates
|American crow||Brown Rat||Eastern Gray Squirrel||Northern Raccoon||Sora rail|
|Biotic||insects, angiosperm seeds||insects, angiosperms seeds||insects, angiosperm seeds||insects, angiosperm seeds||insects, angiosperm seeds|
|Abiotic||soil, water||water, food scraps||water, food scraps||water, food scraps||soil, water|
|Reference:||(Parr, 2005)||(Armitage, 2004)||(Lawniczak, 2002)||(Fox, 2001)||(Stavne, 2002)|
Conclusions: There is a diversity of invertebrates in the transect. Most of the invertebrates found were tiny insects, but were identified by observing their characteristics and using an online key. While no vertebrates were directly observed and it can be hypothesized about what a possible food web could be for this transect. Observing the food web helps understand the different level of systems that are going on in the transect.
Armitage, D. 2004. “Rattus norvegicus”. University of Michigan Museuem of Zoology Animal Diversity Web. (16 Feb. 2015) http://animaldiversity.org/accounts/Rattus_norvegicus/
Fox, R. 2001. "Procyon lotor". University of Michigan Museuem of Zoology Animal Diversity Web. (16 Feb. 2015) http://animaldiversity.org/accounts/Procyon_lotor/
Lawniczak, M. 2002. “Sciurus carolinensis”. University of Michigan Museuem of Zoology Animal Diversity Web. (16 Feb. 2015) http://animaldiversity.org/accounts/Sciurus_carolinensis/
Parr, C. 2005. "Corvus brachyrhynchos". University of Michigan Museuem of Zoology Animal Diversity Web. (16 Feb. 2015) http://animaldiversity.org/accounts/Corvus_brachyrhynchos/
Stavne, R. 2002. "Porzana Carolina”. University of Michigan Museuem of Zoology Animal Diversity Web. (16 Feb. 2015) http://animaldiversity.org/accounts/Porzana_carolina/
2.20.15 Good images of plants. A little repetitive and negative. You could have focussed on describing what you could identify from your plant samples eg. monocot or dicot in the text. A simple google search of cattails can give some more information. SK
Plantae and Fungi: Lab 4 on February 5, 2015
Purpose: The purpose of this lab was to understand the variety of characteristics and diversity of plants. Identifying plants and fungi included methods of observing their appearance and categorizing their method of reproduction.
Materials and Methods:
Electrophersis- 5μL of each of PCR products from the four bacteria samples, which were made last week, were prepared with 3μL of dye. The ladder of the agarose gel was prepared and put into the first lane. After the ladder was put in, each of the four samples were pippetted into the wells (lanes) after it. The agrarose gel was run and the results were observed on a UV table. The best two sequenced bacteria were shipped off to get sequenced.
Plants and Fungi Observation- In this lab, a collection of 5 plant samples from the marsh transect was used. Each sample was observed for vascularization, specialized structures, and mechanism for reproduction. Under a dissection microscope, the plant samples, as well as a mushroom (this was not from the transect) were observed for these characteristics.
Berlese Funnel- A 500g sample of soft soil and leaf litter was gathered from the marsh transect. To make the Berlese Funnel for gathering invertebrates, 25mL of 50:50 ethanol/water solution was added to a 50ml conical tube, which was fixed to a funnel with tape. A black screen was taped to the inside of the bottom of the funnel, and the sample was put on top. The funnel was set in a ring stand and had parafilm hold the base to keep it steady. The funnel was then put under a 40 watt light, 2 inches above the top of the leaf litter. Everything was covered in foil for a week, until the lab next week.
Data Observations: Plants in the Transect- The sample of 5 plants from the transect included four angiosperms and one non-vascular plant, which can be seen in Table 1. The specific species of the plants are still unknown, because it was not possible due to the methods of observation.
Table 1: Characteristics of Plants in the Transect
Sample 1: http://openwetware.org/wiki/Image:800px-IMG_Plant1.JPG Sample 1 In Transect: http://openwetware.org/wiki/Image:800px-IMG_TransPlant1.JPG
Sample 2: http://openwetware.org/wiki/Image:800px-IMG_Plant2.JPG Sample 2 In Transect: http://openwetware.org/wiki/Image:600px-IMG_TransPlant2.JPG
Sample 3: http://openwetware.org/wiki/Image:800px-IMG_Plant3.JPG Sample 3 In Transect: http://openwetware.org/wiki/Image:800px-IMG_TransPlant3.JPG
Sample 4: http://openwetware.org/wiki/Image:800px-IMG_Plant4.JPG Sample 4 In Transect: http://openwetware.org/wiki/Image:800px-IMG_TransPlant4.JPG
Sample 5: http://openwetware.org/wiki/Image:800px-IMG_Plant5.JPG Sample 5 In Transect: http://openwetware.org/wiki/Image:800px-IMG_TransPlant5.JPG
Conclusions: The specific species of plants from the marsh transect are still unknown. Since it is the winter, it was hard to determine if plants were flowering, because most of the plants were dead.
2.9.15 Very good notebook entry. Well structured, relevant images. SK
Microbiology and Identifying Bacteria with DNA Sequences: Lab 3 on January 29, 2015
Purpose: The purpose of this lab was to understand the characteristics of bacteria and how they lived in our ecosystem. Bacteria was obtained from the transect to be viewed under a microscope. Additionally, to further differentiate the different bacterium, staining techniques were used to determine whether or not the bacteria was resistant to antibiotics.
Materials and Methods: Before examining any bacteria, the Hay Infusion Culture was observed for the last time looking for changes in smell and appearance. After examining the Hay Infusion Culture, the eight agar plants of bacteria were observed (four of which had antibiotics and the other did not). Once the number of colonies was calculated for each plate, the plates were used to make wet mounts to put under the microscope to observe more specific characteristics of the bacteria.
Next, the gram straining procedure was done to determine whether if the bacteria were antibiotic resistant, or gram positive. Being gram positive means that the bacteria have a thick layer of peptidoglycan in their cell walls, which will turn purple with staining. Gram negative, however, means that the bacteria have less peptidoglycan, which will turn pink after staining. The gram stain started with a sterilizing a loop with a flame to scrape up a tiny amount of the growth from the surface of the agar. The loop was mixed into a little water on the slide. It was then heat fixed, with the bacteria facing, by moving through the flame 3 times which would dries the water. The bacterial smear was then covered with Gram's iodine mordant for one minute and then rinsed off with water. 95% was then poured over the bacterial smear for about 10-20 seconds. Then, the bacterial smear was soaked with safranin stain for about 20-30 seconds and then was rinsed with water. Once the slide was dry, the slide was viewed at 40X and then at 100X oil immersion.
The last step was setting up a PCR for 16s sequencing. A single colony of bacteria was placed in a sterile tube that contained 100μl of water, which was then incubated at 100°C for 10 minutes. After being incubated, the sample was then centrifuged for 5 minutes at 13,400rmp. Following centrifugation, 5μl of supernatant was placed in the sample tube that had the PCR bead and the 20μl of the primer.
Data Observations: On the final observation of the Hay Infusion Culture, a significant amount of water was evaporated, causing the water to turn more brown. The smell became faint and less foul, which was most likely due to the decrease of water and organisms that have died. When examining the 8 agar plants, the last dilution without the tetracycline had the most colonies grow. Table 1, below, shows the calculated colonies for each plate.
The plates without antibiotics formed more colonies than the plates with the antibiotic. This shows that the bacteria living in the Hay Infusion were not resistant to antibiotics. The colonies on the plates without antibiotics were also much smaller than the colonies on the plates containing antibiotics, and had fewer fungi colonies growing as well. The results of these characteristics and other characteristics of the bacteria can be found in Table 2, below.
Conclusions and Future Observations: In conclusion, there are various types and amounts of bacteria living in our transect, some of which are antibiotic resistant. This is due simply because some colonies of bacteria grew on the agar plates that had tetracycline antibiotic on it.
2.4.15 Very good lab entry. Well laid out. Included detailed description of location of protists in Hay infusion. Gave detailed observations of protists and identified 2 protists. Included images. SK
Identifying Algae and Protist: Lab 2 on January 28, 2015
Purpose: The purpose of this was to understand how unknown organisms can be identified by using a dichotomous key after observing the color, motility, shape, and size. This was used to understand the characteristics of algae and protists, unicellular eukaryotes. Algae and protists will be identified from the Hay Infusion Culture from transect 1 that was made in last week's lab.
Materials and Methods: First, wet mounts of known organisms were observed under the microscope to practice using the dichotomous correctly. It was important to be familiar with how the dichotomous key before successfully identifying organisms in the Hay Infusion. Only after two organisms were successfully identified, the dichotomous key was then able to identify unknown organisms in the Hay Infusion Culture. Before doing that, the culture itself had to be observed and analyzed without any disturbances. After observing the culture, two samples from two different niches (the top and bottom of the Hay Infusion) were taken for observation. A wet mount was made for each sample to observe the different algae and protists. To prepare for Lab 3 for next week, eight petri dishes were prepared, four of which contained agar and tetracycline, whereas the other four will just contain agar.
Data Observations: The Hay Infusion Culture for transect 1 was a brown liquid. It had a gelatinous film on the top with soil on the bottom and crushed leaves scattered throughout, as well on the sides. It had an opaque moist smell--like a wet woodland. The first wet mount was made from the top Hay Infusion on the film surface, not located by any leaves. The organisms that were found in this niche were Colipidium and Chlamydomas. The Colipidium was roughly 75μm, colorless, had an oval-like shape body, and exhibited motion. The Chlamydomas was a single greenish cell about 7.5μm, had an oval-shaped body, and had two observed locomotor flagella.The organism that was found in the second niche, or the bottom of the Hay Infusion with soil and leaves, was the Eudorina. The Eudorina is a colorless spherical colony of about 32 cells about 25μm long. If the Hay Infusion "gew" for another two months, the brown water would slowly evaporate, which would cause the carrying capacity of the organisms to decline. It might even get to the point that the organism can no longer live in their niche, because of limit resouces.
Conclusions and Future Observations: Just by observing the Hay Infusion Culture, it can be seen that many organisms live in different organism in a transect. While only protists and algae were identified this week, these unicellular eukaryotes are not the only organisms living in the Hay Infusion environment. Next week in lab, bacteria will be observed in the petri dishes that were created at the end of this lab.
--Sophia C Zbesko 13:49, 28 January 2015 (EST)
1.27.15 Excellent first entry. Nice format and pictures. Could include a little more detail, but generally very good. SK
Biological Life at AU: Lab 1 on January 15, 2015
Purpose: The purpose of this lab was to analyze and observe a specific transect (an ecosystem) at American University, as well as view evolution through natural selection. A sample from the assigned transect, with biotic and abiotic components, will be obtained and used to examine over the next several weeks of the semester. The sample will be used to investigate the interactions between the different components and analyze all the organisms that lived in that specific niche.
Materials and Methods: First, the TA formed groups of three students who she allocated to each transect. Once assigned, my group went to our transect to observe and analyze it, as well as take a sample with a 50mL conical tube. The tube contained 50% of the soil from the transect itself and the other 50% consisted of several different types of leaves. The contents of the tube were then used to be a Hay Infusion Culture. The Hay Infusion was created by blending 10 grams of the sample, 500mLs of the deer park water, and 0.1mg of dried milk. This mixture was covered with a lid and mixed for 10 seconds and then was then left in the back of the lab without a lid.
Data and Observation: The assigned 20 by 20 foot transect, labeled as 1, was located on the North side of campus on the side of Kogod that faces the Katzen building. The biotic components were cat tail bushes, weeds, grass, fern, and mass. The abiotic, nonliving, components were rocks, soil, a storm drain, salt, and litter.
Conclusions and Future Directions: Both the biotic and abiotic components in the transect are important for the niche and affect the organisms that live in it. Closely viewing the niche will allow us to identify and study the various species that live in our specific niche. It will also provide evidence of whether or not the storm drain, litter, and salt have any effect on the living organisms. The Hay Infusion Culture that was created will be observed in next week's lab and different species will be observed under the microscope. The Hay Infusion will show how an ecosystem, or transect, has various species, as well as provide further information on how to understand how evolution changes life over time.
--Sophia C Zbesko 13:21, 28 January 2015 (EST)
First Entry: Testing to make sure this works. *Sophia C Zbesko 20:48, 25 January 2015 (EST)